The ribosome plays a central role in the conversion of genotype into phenotype in all forms of life. Since core ribosomal functions are highly conserved, the broad, long-term objective of the proposed research is to better understand the structure and function of the ribosome in the simplest free-living organisms, bacteria. Ribosomes from the bacterium Escherichia coli have been studied genetically, biochemically, and structurally for over four decades. Thus, although preliminary x-ray crystal structures have been solved of the bacterial ribosome from a poorly characterized thermophile, structures of the E. coli ribosome will bring significant advances to our understanding of protein synthesis. The ribosome works by means of specific initiation, elongation, and termination steps. During elongation, the ribosome must read the genetic code in messenger RNA (mRNA) for each successive amino acid that needs to be added to the growing polypeptide chain, a process termed decoding. Ribosomal decoding of mRNA will be probed by both structural and biochemical approaches. The specific aims are: 1) to improve the resolution of diffraction from E. coli ribosome crystals obtained in my laboratory by forming a ribosome complex that mimics decoding, 2) to solve the structure of the ribosome in the decoding complex, and 3) as a model for decoding defects, to examine the mechanism of stop codon read-through, or nonsense suppression, caused by mutations in tRNA. These experiments will advance our knowledge of how genetic information is converted into the working parts of the cell. They will provide a structural and biochemical foundation for modeling the molecular basis for ribosome function in protein synthesis. Insights from these studies will also lead to a clearer view of steps in protein synthesis that are inhibited by many classes of antibiotics.